Expression 1680 scanner

Proteins were separated based
on their apparent molecular weight using a modified version of the
Laemmli SDS-PAGE^{17 (link)} with a 5% for stacking
and 10% separating gel. 10 μg of protein from each sample was
loaded alongside a molecular-weight standard (Precision Plus Protein
Kaleidoscope, Bio-Rad). Gels were run at 94 V and were stopped when
the dye front reached ∼1 cm from the bottom.
Following
electrophoresis, the separating gel was diffusion stained (45.5% methanol,
45.5% RO H₂O, 9.0% acetic acid, and 0.25% Coomassie Brilliant
Blue R250) and gently agitated overnight. The next day, the gel was
destained twice with destain 1 (45.5% methanol, 45.5% RO H₂O, and 9.0% acetic acid) in 3 h intervals with agitation. Then, the
gel was placed in destain 2 overnight and agitated. Finally, the gel
was stored in 7.5% acetic acid for 1 day and scanned the following
day with an Epson Expression 1680 Scanner (Version 3.04A, Professional
Mode, Reflective, Photo Auto Exposure type, 24-bit color and 8-bit
gray-scale, Best Scanning Quality, dpi 1200, gamma 1.58).

The auxin transport inhibitor TIBA (2, 3, 5-triiodobenzoic acid; Sigma Aldrich) was dissolved in absolute ethanol and filter sterilised TIBA was added to sterilized half-strength liquid Hoagland medium to give a final concentration of 10 μM. TIBA treatment was conducted on five-day-old lupin seedlings by transferring the seedlings to half-strength hydroponic Hoagland medium containing 10 μM TIBA (+TIBA). For controls (−TIBA), the seedlings were transferred to the half-strength hydroponic Hoagland medium with the same amount of ethanol as in + TIBA medium. 48 h after treatments, the roots were inoculated by placing a 4 mm diameter plug of P. cinnamomi mycelium at the root tips and seedlings were kept in half-strength hydroponic Hoagland medium until harvested.
A minimum of 10 lupin roots per treatment were assessed for lesion development and the experiment was repeated twice. The level of infection was measured 48 and 72 h after P. cinnamomi inoculation and the data were presented as mean percentage infected root area. The digital images of the roots were captured with as Epson Expression 1680 scanner and the area of lesions formed by P. cinnamomi infection were calculated using the program WinRHIZO™ (Régents Instruments, Inc.).

After 7 days, plants were washed with sterile distilled water, and dried with bibulous paper to measure the fresh weights (FW). Plant materials were incubated at 105 °C for 15 min, placed at 70 °C for 72 h and then weighted to determine the dry weights (DW). Fifteen plants were measured for each treatment. The area of the fully expanded cucumber leaves was estimated using an Expression 1680 scanner (Epson, Sydney, Australia) and analyzed with WinRHIZO (Regent Instruments Ltd, Ontario, Canada).
Chlorophyll content in cucumber leaves was extracted with a mixture of acetone, ethanol, and water (4.5: 4.5: 1, V:V:V) and analyzed according to the method of Arnon^{16 (link)}. The fourth leaves were used for net photosynthetic rate (Pn) analysis with a portable photosynthesis system (LI-6400, LI-COR Inc., Lincoln, USA), at a temperature of 25 °C, 85% relative humidity, a cuvette air flow rate of 500 mL min⁻¹, and an ambient CO₂ concentration of 380 μmol mol⁻¹. A PPFD of 600 μmol m⁻² s⁻¹ was provided by a mixture of red and blue light-emitting diodes.